Every multicellular organism starts its development by fertilization and is completed as an individual having various tissues and well-balanced system by undergoing cell division (cleavage) and cell differentiation. The differentiation process is highly complicated and is thought that important interactions between cells called induction phenomena takes place in many steps of the differentiation stratum. The elucidation of “molecule that dominates morphogenesis” is said to be the most significant. Amphibian embryos are often and mostly used as materials for these studies, nevertheless, the basic rule of body formation is common to all the vertebrates and homologous genes are known to have quite a similar function even among different species.
Amphibian embryo has conventionally been regarded as an extremely valuable material in the field of experimental embryology with which many studies have been made. This is because amphibian egg fertilizes and develops externally, its large egg makes embryo operation possible, and its time course changes can easily be observed. The amphibian blastopore upper lip of gastrula is a special region and when it is transplanted into the ventral side of another embryo, a secondary embryo including head or body-tail part is induced. This is why the blastopore upper lip is named “organizer” as a region that acts as the center of morphogenesis that determines the embryo system. It is well known that the organizer induces central nerve by functioning to presumptive ectoderm during invagination of the primitive gut, while the organizer itself differentiates into dorsal mesoderm and anterior endoderm.
On the other hand, pancreas is an endocrine organ which indicates histomorphology and manner of development that is common to most vertebrates, namely, mammals, birds, reptiles and amphibians, and is an exocrine organ as well. It is known that during the developmental process, dorsal and ventral primordia arise from the endoderm, and they fuse to be pancreas (Development 121, 1569-1580, 1995).
It has been said that there exists a mesoderm in the vicinity of endoderm in the process of embryogenesis, and that action from mesenchyme to endoderm is necessary for the differentiation of pancreas (Dev. Biol. 4, 242-255, 1962). Recent studies have reported that involvement of notochord is required for pancreatic formation of chick, and that notochord suppresses the Shh expression in the endoderm in its vicinity to differentiate pancreas. It has also been reported that it is the endoderm of the pancreas presumptive region that differentiates into pancreas by action of notochord, and that differentiation into pancreas is not found in endoderm aside from the pancreas presumptive region, even when notochord coexists (Development 124, 4243-4252, 1997, Proc. Natl. Acad. Sci. USA 95, 13036-13041, 1998, Genes and Dev. 12, 1705-1713, 1998).
Further, from research at gene level, it had been reported that homeobox gene, known as ipf-1 and pdx-1 that express in the pancreatic primordium of mouse, is essential to the formation process of pancreas. Gene targeting experiment of ipf-1 revealed that mouse embryo without this gene was pancreas-defective (Nature 371, 606-609, 1994). However, the primordium of pancreas was formed even when this gene was deficient, and the existence of glucagon-positive cells was detected (Development 122, 983-995, 1996). In addition, it is known that the vegetal pole cell of Xenopus blastula expresses both of XlHbox8 that is a pancreas-specific transcription factor and IFABP that is the homolog of PDX-1 and a small intestinal epithelium marker, however, when the signal of TGF-β system at the endoderm is inhibited, the expression of XlHbox8 is inhibited (Development 122, 1007-1015, 1996).
On the other hand, it is known that retinoic acid is a regulatory factor for the embryonic patterning along the anteroposterior axis (Nature 340, 140-144, 1989, Development 112, 945-958, 1991, Dev. Biol. 192, 1-16, 1997, Zool. Sci. 15, 879-886, 1998), and that this retinoic acid transforms anterior neural tissue of Xenopus embryo to a posterior one and is effective on mesodermal development (Genes Dev. 5, 175-187, 1991, Develop. Growth. Differ. 35, 123-128, 1993). It has also been reported that treatment with activin induces most mesodermal tissues such as notochord, muscle, mesenchyme and coelomic epithelium, dose-dependently in Xenopus animal cap cells (Roux's Arch. Dev. Biol. 198, 330-335, 1990, Nature 347, 391-394, 1990, Roux's Arch. Dev. Biol. 200, 230-233, 1991). Changing the dosage of retinoic acid that is co-treated with activin enables the mesodermal tissues such as notochord, muscle and pronephros that differentiate from animal cap cells to be lateralized and posteriorized (Develop. Growth. Differ. 35, 123-128, 1993).
As to the action of retinoic acid to the endodermal organ, it has been reported by Dixon et al. that when Xenopus embryos at developmental stage 22 to 32 are treated with retinoic acid, the morphology of the digestive organs such as the intestines, liver and stomach become abnormal, nevertheless, it has also been reported that pancreas of Xenopus embryos at developmental stage 22 to 32 that had been treated with retinoic acid is formed normally, and no effect is found in the expression of XlHbox8, an endoderm-specific marker (Dev. Genes Evol. 208, 318-326, 1998).
To date, specific induction of a specific organ in vitro had been regarded as being extremely difficult, and the complex differentiation and formation mechanisms of pancreas remain unclear. The object of the present invention is to provide a method, whereby a pancreas induced in vitro which enables to obtain findings on the differentiation and formation mechanisms of pancreas and thus is useful in developmental engineering or organ engineering, a pancreas for transplantation by which it can be evaluated whether or not a pancreas induced in vitro can function in practice in vivo, and a pancreas induced in vitro which contributes to the development of diagnosis and treatment of pancreatic diseases of higher animals, can be artificially and efficiently induced from a gastrula excluding the presumptive region of pancreas.